Glutamate mediates rapid synaptic responses in many parts of the vertebrate nervous system. While activation of the AMPA subtype of glutamate receptor requires a high concentration of transmitter, these receptors desensitize to low concentrations, a process which contributes significantly to determining the amplitude and decay time of synaptic responses. Additionally, the NMDA and metabotropic glutamate receptors are activated by low concentrations of transmitter. As the only known mechanism for removing glutamate from the extracellular space is uptake by pumps, glutamate could diffuse from the synaptic cleft, influencing the activity of surrounding synapses by binding to such high affinity sites. In order to understand how the transmitter could have such widespread effects, we must answer several questions. How much glutamate is released? How far can it diffuse from the synapse? What are its actions outside the cleft" Do glutamate pumps limits its sphere of influence and its lifetime? The neurons in the chick nucleus magnocellularis are well suited to studying these issues. The neurons are adendritic and receive several large excitatory axon terminals, as well as inhibitory boutons, on the cell body. These inputs can be stimulated separately. Glutamate released by the excitatory synapse may require tens of milliseconds to be removed. We will use the these cells in slices and as dissociated cells in the proposed experiments. Glutamate concentration in the synaptic cleft will be assayed by a novel, empirical method which takes advantage of voltage-sensitive channel kinetics of the AMPA receptor and the dependence of those kinetics on concentration. Glutamate outside the synaptic cleft will be detected cy the calibrated response of receptor-rich membrane patches positioned near the synapse. Additionally, we will determine the biological actions of diffusing transmitter on adjacent excitatory and inhibitory synapses. The role of glutamate uptake in regulating the lifetime of glutamate in the cleft and outside the cleft will be assayed. The site of uptake in this system, whether pre- or postsynaptic or glial, will be determined. Finally, the buffering of the transmitter by binding to receptors will be explored as it relates to controlling the lifetime of the transmitter in the cleft.